PROJECT SUMMARY/ABSTRACT Ewing sarcoma (ES) is the second most common pediatric tumor involving bone. Survival rates for patients with metastatic disease remain dismal at 20-30%, presenting an unmet clinical need. Pediatric cancers, on average, exhibit lower mutational burdens compared to adult cancers, concealing disease biology and potential targets for treatment, posing a major barrier to novel drug discovery. Indeed, ES tumors exhibit extremely few oncogenic mutations, with the exception of the characteristic 11;22 chromosomal translocation, which creates the oncogenic EWS-FLI fusion transcription factor in 85-90% of cases. The EWS-FLI oncoprotein closes and opens chromatin at specific motifs in the genome, inducing profound epigenetic dysregulation. To investigate ES biology, a genome-scale CRISPR-Cas9 screen was performed, which identified the transcription factor, ETV6, as a gene essential for ES cell survival. ETV6 is a repressive transcription factor that has previously been shown to mediate gene repression through the direct recruitment of the histone deacetylating (HDAC) enzyme, HDAC3. Preliminary experiments have demonstrated that ETV6 shares thousands of genomic binding sites with EWS- FLI in ES cells, including binding at EWS-FLI-up-regulated genes. Furthermore, ETV6 over-expression reduces abundance of the promotive histone mark, H3K27ac, consistent with its previously described repressive function. Thus, this proposal will address the hypothesis that ETV6 is essential for ES cell survival by repressing a subset of EWS-FLI-induced target genes via the recruitment of HDAC3. Aim 1 will assess the essentiality of this gene in models of ES in vitro and in vivo and elucidate the molecular mechanism of decreased survival of ES cells when ETV6 is suppressed. Aim 2 will define the transcriptional programs of ETV6 and EWS-FLI to determine whether ETV6 represses a subset of EWS-FLI target genes that are deleterious for cell survival and growth. Finally, Aim 3 will investigate whether ETV6 represses EWS-FLI target genes by recruiting HDAC3 and whether ES cells are sensitive to the selective HDAC3 inhibitor, RGFP966. Altogether, this work will characterize a novel essential gene in ES and highlight a potential novel therapeutic approach for ES.